Characterization of the P2 receptors on the human umbilical vein endothelial cell line ECV304

Abstract

To characterize the P2 receptors present on the human umbilical vein endothelial‐derived cell line, ECV304, cytosolic Ca 2+ , ([Ca 2+ ] c ), responses were recorded in single cells and in cell suspensions to a series of nucleotides and nucleotide agonists. Concentration response curves were obtained in fura‐2‐loaded ECV304 cell suspensions, with EC 50 values of 4.2 μ M for ATP, 2.5 μ M for UTP and 14 μ M for adenosine‐5′‐ O ‐(3‐thio)triphosphate (ATPγS). EC 50 values for 2‐methylthioATP, ADP, adenosine‐5′‐ O ‐(2‐thio)diphosphate (ADPβS) and AMP were 0.5 μ M , 3.5 μ M , 15 μ M and 4.7 μ M respectively, but maximal [Ca 2+ ] c responses were less than those produced by a maximal addition of ATP/UTP. ECV304 cells were unresponsive to UDP and β,γ,methyleneATP. Cross‐desensitization studies on ECV304 cells suggested that ATP and UTP recognized the same receptor. However, ADP recognized a receptor distinct from the UTP‐sensitive receptor and AMP recognized a third distinct receptor. ECV304 [Ca 2+ ] c responses to 2‐methylthioATP were inhibited in the presence of 30 μ M pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), whereas [Ca 2+ ] c responses to UTP were unaffected by this treatment. ECV304 cells responded to the diadenosine polyphosphate Ap 3 A with rises in [Ca 2+ ] c . Apparent responses to Ap 4 A, Ap 5 A and Ap 6 A, were shown to be due to a minor nucleotide contaminant that could be removed by pre‐treatment of the diadenosine samples with either alkaline phosphatase or apyrase. ECV304 cells display a pharmacology consistent with the presence of at least two P2 receptors; a P2Y 2 receptor insensitive to the diadenosine polyphosphates and a P2Y 1 receptor sensitive to Ap 3 A. In addition, ECV304 cells respond to AMP with increases in [Ca 2+ ] c via an as yet uncharacterized receptor. British Journal of Pharmacology (1998) 125 , 357–364; doi: 10.1038/sj.bjp.0702082